The Seminar, part of a series which aims to stimulate scientific discussion at UKZN, was attended by about 100 students and researchers from UKZN and abroad.

Daum’s talk titled: “Characterisation of the PncA Gene from MTB Isolates and Sputum Specimens Using Next-Generation Sequencing”, outlined the use of DNA sequencing to study tuberculosis (TB) drug resistance.

TB is a debilitating disease caused by mycobacterium tuberculosis and is treatable but recently, drug resistant, multi-drug resistant and extensive drug resistant strains of MTB have been identified in an increasing number of patients. South Africa has one of the highest TB disease burdens worldwide.

DNA sequencing is the process of determining the precise nucleotide sequences in genetic regions, or entire genomes of organisms such as TB.

Daum explained the use of Next Generation Sequencing (NGS), a third generation technology that allows for massively parallel sequencing of DNA to identify mutations that confer resistance to anti-TB drugs currently in use. He focused on the pncA gene which encodes pyrazinamidase in mycobacterium (MTB) species, the gene that is associated with resistance to Pyrazinamide (PZA), a drug used to treat TB.

Identifying drug resistance in patients infected with TB often takes several weeks but Daum has developed a standardised sequencing methodology to identify mutations conferring resistance to several anti-TB drugs within days of sample collection.

He spoke about the use of next generation sequencing in his study of the PncA gene, (encoding Pyrazinamidase) from isolates collected from patients in Pretoria. Samples were transferred to the PrimeStore MTM™ tubes and shipped from Pretoria to Texas where he used the Ion Torrent (Life Technologies) to sequence the pncA gene.

Using computational tools he identified known mutations conferring resistance to PZA and together with clinical data was able to infer the incidence of drug resistant strains in the cohort. In nine of 26 isolates, pncA resistance was identified.

Daum illustrated the power of next generation sequencing when he explained how he used NGS to identifiy mutations in three patients during from a Milwaukee (United States) outbreak. Patient samples were tested using conventional GeneXpert TB/Rif assay and also sequenced. Two of the three patients were infected with a multi-drug resistant strain of TB, while the remaining patient was infected with a drug susceptible strain of TB according to the GeneXpert assay but actually had drug resistance mutations to isoniazid, a first line TB drug which was detected only though NGS.

With the turnaround time of five days for analysis to report results, Daum said this showed that next generation sequencing was the way forward for health care since it provided detailed resistance information within a short space of time.

Finally, Daum illustrated that this protocol was robust and very sensitive by showing the audience sequence data from TB isolates collected in the early 1990s. Sequencing revealed that XDR TB may have been present in the community far earlier than was originally estimated. His research also revealed novel mutations that would have been missed had it not been sequenced using NGS technology.

Daum highlighted the power of genomics in that sequencing was able to detect mutations and resistance patterns that conventional resistance testing did not identify. ‘These results are very interesting and advance our understanding of the origin and spread of TB drug resistance in South Africa. It also shows how genomics technologies are quickly evolving,’ said Professor Tulio de Oliveira.

De Oliveira is attached to UKZN’s Africa Centre for Health and Population Studies and College of Health Sciences, and is the Program Director (Genomics) at the Wellcome Trust Africa Centre for Health and Population Studies, Mtubatuba, South Africa. ‘We collaborate with Dr Daum as part of a pilot project to evaluate the utility of PrimeStore in sequencing TB genomes directly from sputum sampled in rural KwaZulu-Natal. Our objective is to be able to sample and genotype TB after a few days from collection. If this project is successful, it will allow the Africa Centre and its partners, such as K-RITH and CAPRISA, to be able to produce large scale TB genotypes to answer scientific questions related to transmission and drug resistance development in KwaZulu-Natal.’

* Daum, the Chief Scientific Officer for Longhorn Vaccines and Diagnostics at the University of Texas San Antonio, previously worked as a Civilian Scientist for the United States Air Force Institute for Operational Health (AFIOH). He possesses extensive experience with respiratory pathogens in molecular epidemiology and the development of pathogen diagnostics. He has published 17 peer reviewed scientific papers, has presented over 30 scientific abstracts and lectures, and holds two patents.